Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.

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It has come to our attention that in preparing the final version of this paper, we inadvertently misspelled the first name of an author Ziming Zhou as ''Zimin Zhou''. In addition, we have made two errors in describing the reagents in the STAR Methods. First, under the subheading of ''Synthesis of barcoded beads'' in the Method Details section, the supplier of the magnetic beads coated with carboxyl groups should be Suzhou Knowledge & Benefit Sphere Tech. Co., Ltd. (diameter 20-25 mm, http://www.kbspheretech. com/), instead of Zhiyi. Second, under the subheading of ''Cell collection and lysis'' in the Method Details section, the concentration of Tris-HCL for the cold lysis buffer should be 0.1 M, instead of 1 M. These errors have been corrected online, and we apologize for any confusions we may have caused.

Embryonic stem cell (ES cell) lines were first generated by culturing mouse inner cell mass (ICM) on feeder layers in 1981 [1]. However, in large domestic animals, attempts to establish ES cell lines from ICM of blastocysts or the later epiblast have not been successful. This has hindered the efficient production of genetically modified livestocks by using ES-based approaches. Recently, it was found that ectopic expression of various combinations of transcription factors is able to reprogram somatic cells to a pluripotent state [2][3][4][5]. These induced pluripotent stem (iPS) cells show similarities to embryo-derived ES cells and can be used to produce viable mice through tetraploid complementation [6,7]. So far, iPS cells of several mammalian species have been successfully generated [2,3,[8][9][10][11][12]. In this letter, we report the first establishment of bovine iPS cells using defined transcription factors and a modified culture medium.cDNAs coding for the bovine OCT4 (also named POU5F1), SOX2, KLF4, MYC, LIN28, and NANOG genes were cloned into pMXs retroviral vector. The pMXs plasmids containing human OCT4, SOX2, KLF4, and c-MYC genes were all purchased from Addgene. GP2-293 cells were used as the packaging cell line for retroviral production. Bovine fibroblasts used in this study were derived from an E55 Western Shandong Yellow cattle fetus. Three sets of factors, termed b4TF, b6TF, and h4TF, were used to transduce cells by overnight retroviral infection, respectively. Whereas the former two included only bovine factors (b4TF: bOCT4, bSOX2, bMYC, bKLF4; b6TF: bOCT4, bSOX2, bKLF4, bMYC, bLIN28, bNANOG), the latter employed only human factors (hOCT4, hSOX2, hc-MYC, hKLF4). On day 2, the infected cells were harvested by trypsinization and plated onto mitomycin C-treated MEF feeders at a density of 1 × 10 4 cells per 100-mm dish. The next day after being seeded onto feeders, growing cells were cultured in iPS media ( Figure 1A and Supplementary information, Table S1). Bovine iPS (hereinafter named biPS) cells with a mouse ES-like morphology were detected after ~3 Figure 1B). On day 21-35, colonies were isolated mechanically using a 200 µl pipette and transferred to feeder-coated tissue culture dishes. The biPS cells were split with trypsin at a ratio of 1:10 every 4-5 days afterwards (Supplementary information, Data S1). A total of 26 b6TF-derived colonies have been expanded into biPS cell lines. These lines maintained good ES-like morphology for more than 16 passages. However, none of the colonies generated by b4TF or h4TF could be passaged more than six times. Importantly, we showed that the combination of six transcription factors (b6TF set) significantly increased the efficiency of iPS cell generation (by three-fold) compared to the other two combinations ( Figure 1C).We tested eight different types of biPS culture media (Supplementary information, Table S1) by assessing the numbers of ES-like colonies obtained from b6TF-transduced BEFs on day 28. Three out of the eight media could efficiently generat...